The invention relates to the diagnosis and treatment of patients at risk for methionine synthase deficiency and associated altered risk for diseases such as neural tube defects, cardiovascular disease, and cancer.
Methionine synthase (EC 2.1.1.13, 5-methyltetrahydrofolate-homocysteine methyltransferase) catalyses the remethylation of homocysteine to methionine in a reaction in which methylcobalamin serves as an intermediate methyl carrier. This occurs by transfer of the methyl group of 5-methyltetrahydrofolate to the enzyme-bound cob(I)alamin to form methylcobalamin with subsequent transfer of the methyl group to homocysteine to form methionine. Over time, cob(I)alamin may become oxidized to cob(II)alamin rendering the enzyme inactive. Regeneration of the functional enzyme occurs through the methionine synthase-mediated methylation of the cob(II)alamin in which S-adenosylmethionine is utilized as methyl donor. In E. coli, two flavodoxins have been implicated in the reductive activation of methionine synthase (Fujii, K. and Huennekens, F. M. (1974) J. Biol. Chem., 249, 6745-6753). A methionine synthase-linked reducing system has yet to be identified in mammalian cells.
Deficiency of methionine synthase activity results in hyperhomocysteinemia, homocystinuria, and megaloblastic anemia without methylmalonic aciduria (Rosenblatt, D. S. (1995) The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York, pp. 3111-3128; Fenton, W. A. and Rosenberg, L. E. (1995) The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York, pp. 3129-3149). Two classes of methionine synthase-associated genetic diseases have been proposed based on complementation experiments between patient fibroblast cell lines (Watkins, D. and Rosenblatt, D. S. (1988) J. Clin. Invest., 81, 1690-1694). One complementation group, cblE, has been postulated to be due to deficiency of the reducing system required for methionine synthesis (Rosenblatt, D. S., Cooper, B. A., Pottier, A., Lue-Shing, H., Matiaszuk, N. and Grauer, K. (1984) i J. Clin. Invest., 74, 2149-2156). Cells from patients in the cblE group fail to incorporate 14C-methyltetrahydrofolate into methionine in whole cells but have significant methionine synthase activity in cell extracts in the presence of a potent reducing agent. The second complementation group, cblG group, is thought to result from defects of the methionine synthase apoenzyme. Mutant cells from this group show deficient methionine synthase activity in both whole cells and cell extracts (Watkins, D. and Rosenblatt, D. S. (1988) J. Clin. Invest., 81, 1690-1694; Watkins, D. and Rosenblatt, D. S. (1989) Am. J. Med. Genet., 34, 427-434). Moreover, some cblG patients show defective binding of cobalamin to methionine synthase in cells incubated with radiolabelled cyanocobalamin (Sillaots, S. L., Hall, C. A., Hurteloup, V., and Rosenblatt, D. S. (1992) Biochem. Med. Metab. Biol., 47, 242-249).
The cobalamin-dependent methionine synthase of E. coli has been crystallized and the structure of its active site determined (Luschinsky, C. L., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1992) J. Molec. Biol., 225, 557-560; Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994) Science, 266, 1669-1674.). The gene encoding methionine synthase has not been cloned from mammals.
We have cloned a gene for mammalian methionine synthase from humans and discovered that mutations in this gene are associated with hyperhomocysteinemia. Hyperhomocysteinemia is a condition that has been implicated in cardiovascular disease and neural tube defects. The presence of such mutations in methionine synthase gene are, thus, associated with increased risk for cardiovascular disease, altered risk for neural tube defects, and decreased risk of colon cancer. The invention features methods for risk detection and treatment of patients with hyperhomocysteinemia, cardiovascular disease, neural tube defects, and cancer. The invention also features compounds and kits which may be used to practice the methods of the invention, methods and compounds for treating or preventing these conditions and methods of identifying therapeutics for the treatment and prevention of these conditions.
In the first aspect, the invention provides purified wild-type mammalian methionine synthase gene, and mutated and polymorphic versions of the mammalian methionine synthase gene, fragments of the wild-type, mutated, and polymorphic gene, and sense and antisense sequences which may be used in the methods of the invention. Preferably, the gene is human. The proteins encoded therefrom are also an aspect of the invention as is a methionine synthase polypeptide having conservative substitutions. Preferably, the protein is a recombinant or purified protein having a mutation conferring hyperhomocysteinemia when present in a mammal. In addition, nucleic acids, including genomic DNA, mRNA, and cDNA, and the nucleic acid set forth in SEQ ID NO: 1, or degenerate variants thereof, are provided. The shorter nucleic acid sequences are appropriate for use in cloning, characterizing mutations, the construction of mutations, and creating deletions. In one embodiment, the nucleic acid set forth in SEQ ID NO: 1 is a probe that hybridizes at high stringency to sequences found within the nucleic acid of SEQ ID NO: 1. In further embodiments, the probe has a sequence complementary to at least 50% of at least 60 nucleotides, or the sequence is complementary to at least 90% of at least 18 nucleotides. Protein fragments also are provided. The shorter peptides may be used, for example, in the generation of antibodies to the methionine synthase protein. In some embodiments of this aspect of the invention nucleic acid fragments useful for detection of mutations in the region of the methionine synthase gene which encodes the cobalamin binding domain, and for detecting those mutations which indicate an increased likelihood of hyperhomocysteinemia, are preferred. Most preferred fragments are those useful for detecting the 2756 Axe2x86x92G, xcex94bp 2640-2642, and 2758 Cxe2x86x92G mutations/polymorphisms. Given Applicants"" discovery, one skilled in the art may readily determine which nucleic acids, detection methods, and mutations are most useful. Mutant proteins encoded by these mutations, including, but not limited to, H920D, xcex94Ile 881, and D919G are also provided by the invention (see, for example SEQ ID NOs: 74 and 75). Such mutant and polymorphic polypeptides may have decreased or increased biological activity, relative to wild-type methionine synthase.
In a related aspect, the invention provides antibodies that specifically bind mammalian methionine synthase, and a method for generating such an antibody. The antibody may specifically bind a wild-type methionine synthase, or a mutant or polymorphic methionine synthase. A method for detecting a wild-type, mutant, or polymorphic methionine synthase using the antibody is also provided by the invention.
In a second aspect, the invention provides a method for detecting an increased or decreased risk for hyperhomocysteinemia in a fetus or individual patient. Such a fetus or patient is at increased or decreased risk for neural tube defects and/or cardiovascular disease and at a decreased risk of developing colon cancer. The method includes detection of mutations in the methionine synthase gene present in the fetus, the individual patient, and/or the blood relatives of the fetus and patient. The presence of mutations, particularly in the cobalamin binding domain, indicate an altered (e.g., increased or decreased) risk of hyperhomocysteinemia, neural tube defects, cancer, and cardiovascular disease.
In a related aspect, the invention provides kits for the detection of mutations in the human methionine synthase gene. Such kits may include, for example, nucleic acid sequences, including probes, useful for PCR, SSCP, or RFLP detection of such mutations. Antibodies specific for proteins having mutations, correlated with an increased likelihood of hyperhomocysteinemia, may also be included in the kits of the invention.
In a fourth aspect, the invention features a method for screening for compounds which alter methionine synthase expression or ameliorate or exacerbate conditions of hyperhomocysteinemia. In various embodiments, the invention includes monitoring mutant or wild-type mammalian methionine synthase biological activity by monitoring methionine synthase enzymatic activity, or monitoring methionine synthase gene expression levels, by monitoring methionine synthase gene transcription, RNA stability, RNA translation and/or protein stability. In preferred embodiments the methionine synthase gene or protein being monitored is a gene or protein having a mutation associated with hyperhomocysteinemia, and samples are selected from purifed or partially purified methionine synthase, cell lysate, a cell, or an animal. Standard assay techniques known to those skilled in the art may be employed in the various embodiments. Compounds detected using this screen can be used to prevent or treat cardiovascular disease and neural tube defects or, in the alternative, to prevent or treat colon cancer. Kits for performing the above screens are also a part of the invention.
In a related aspect, the invention provides nucleic acids encoding wild-type, polymorphic, and mutated methionine synthase, in which the nucleic acid is operably linked to regulatory sequences, comprising a promoter, for the expression of the encoded polypeptides. In one embodiment, the promoter is inducible. The invention also provides cells, including prokaryotic and eukaryotic cells, comprising the nucleic acids. The eukaryotic cells may be yeast cells or mammalian cells.
In another related aspect, the invention features a transgenic mammal having a methionine synthase transgene. The gene may be wild-type, or may contain a mutation or polymorphism. The mammal may have a mutation associated with hyperhomocysteinemia in its methionine synthase gene in an expressible genetic construction or may have a deletion or knockout mutation in one or both alleles sufficient to abolish methionine synthase expression from the locus. In addition, or as a replacement, the mammal may have the methionine synthase gene from another species. For example, in one preferred embodiment the transgenic mammal is a rodent such as a mouse and the transgene is from a human. Cells from these transgenic or knockout animals are also provided by the invention. Such transgenic mammals may be used to screen for drugs for the treatment of diseases related to hyperhomocysteinemia.
In a sixth aspect, the invention features a method for treating patients with neural tube defects, colon cancer or related cancers by the delivery of antisense methionine synthase nucleic acid sufficient to lower the levels of methionine synthase polypeptide biological activity.
In a related aspect, the invention provides a method for treating or preventing cardiovascular disease, neural tube defects and cancer. The method comprises detecting an altered risk of such defects by analyzing methionine synthase nucleic acid, potential test subjects being a mammal, a potential parent, either male or female, a pregnant mammal, or a developing embryo or fetus, and then by exposing the subject (e.g., patient or pregnant mammal) to metabolites or cofactors such as, but not limited to, folate, cobalamin, S-adenosyl methionine, betaine, or methionine. In another related aspect, the invention features a method of pretreating or treating colon cancer or neural tube defects by inhibiting or activating methionine synthase biological activity in a mammal, pregnant mammal, embryo, or fetus. In preferred embodiments, this inhibiting or activating may be effected by exposing the subject to nucleic acids, peptides or small molecule-based inhibitors or activators of methionine synthase or substrates. The exposure is to quantities of the compound sufficient to reduce the probability of the subject developing the disease or to confer an increased likelihood of a decrease in the disease symptoms of the subject.
By xe2x80x9cmethionine synthase,xe2x80x9d xe2x80x9cmethionine synthase protein,xe2x80x9d or xe2x80x9cmethionine synthase polypeptidexe2x80x9d is meant a polypeptide, or fragment thereof, which has at least 50% amino acid identity to boxes 1-4 of the human methionine synthase polypeptide (SEQ ID NO: 2) (see FIG. 1). It is understood that polypeptide products from splice variants of methionine synthase gene sequences are also included in this definition. Preferably, the methionine synthase protein is encoded by nucleic acid having a sequence which hybridizes to a nucleic acid sequence present in SEQ ID NO: 1 (human methionine synthase cDNA) under stringent conditions. Even more preferably the encoded polypeptide also has methionine synthase biological activity.
By xe2x80x9cmethionine synthase nucleic acidxe2x80x9d or xe2x80x9cmethionine synthase genexe2x80x9d is meant a nucleic acid, such as genomic DNA, cDNA, or mRNA, that encodes methionine synthase, a methionine synthase protein, methionine synthase polypeptide, or portion thereof, as defined above. A methionine synthase nucleic acid also may be a methionine synthase primer or probe, or antisense nucleic acid that is complementary to a methionine synthase nucleic acid.
By xe2x80x9cwild-type methionine synthasexe2x80x9d is meant a methionine synthase nucleic acid or methionine synthase polypeptide having the nucleic acid and/or amino acid sequence most often observed among members of a given animal species and not statistically associated with a disease phenotype. Wild-type methionine synthase is biologically active methionine synthase. A wild-type methionine synthase is, for example, a human methionine synthase polypeptide having the sequence of SEQ ID NO: 1.
By xe2x80x9cmutant methionine synthase,xe2x80x9d xe2x80x9cmethionine synthase mutation(s),xe2x80x9d xe2x80x9cmutations in methionine synthase,xe2x80x9d xe2x80x9cpolymorphic methionine synthase,xe2x80x9d xe2x80x9cmethionine synthase polymorphism(s),xe2x80x9d xe2x80x9cpolymorphisms in methionine synthase,xe2x80x9d is meant a methionine synthase polypeptide or nucleic acid having a sequence that deviates from the wild-type sequence in a manner sufficient to confer an altered risk for a disease phenotype, or enhanced protection against a disease, in at least some genetic and/or environmental backgrounds. Such mutations may be naturally occurring or artificially induced. They may be, without limitation, insertion, deletion, frameshift, or missense mutations. A mutant methionine synthase protein may have one or more mutations, and such mutations may affect different aspects of methionine synthase biological activity (protein function), to various degrees. Alternatively, a methionine synthase mutation may indirectly affect methionine synthase biological activity by influencing, for example, the transcriptional activity of a gene encoding methionine synthase, or the stability of methionine synthase mRNA. For example, a mutant methionine synthase gene may be a gene which expresses a mutant methionine synthase protein or may be a gene which alters the level of methionine synthase protein in a manner sufficient to confer a disease phenotype in at least some genetic and/or environmental backgrounds.
By xe2x80x9cbiologically activexe2x80x9d methionine synthase is meant a methionine synthase protein or methionine synthase gene that provides at least one biological function equivalent to that of the wild-type methionine synthase polypeptide or methionine synthase gene. Biological activities of a methionine synthase polypeptide include, and are not limited to, the ability to catalyze the methylation of homocysteine to generate methionine. Preferably, a biologically active methionine synthase will display activity equivalent to at least 35% of wild-type activity, more preferably, a biologically active methionine synthase will display at least 40-55% of wild-type activity, still more preferably, a biologically active methionine synthase will display at least 60-75% of wild-type activity, and most preferably, a biologically active methionine synthase will display at least 80-90% of wild-type activity. A biologically active methionine synthase also may display more than 100% of wild-type activity. Preferably, the biological activity of the wild-type methionine synthase is determined using the methionine synthase nucleic acid of SEQ ID NO: 1 or methionine synthase polypeptide of SEQ ID NO: 2. The degree of methionine synthase biological activity may be intrinsic to the methionine synthase polypeptide itself, or may be modulated by increasing or decreasing the number of methionine synthase polypeptide molecules present intracellularly.
By xe2x80x9chigh stringency conditionsxe2x80x9d is meant hybridization in 2xc3x97SSC at 40xc2x0 C. with a DNA probe length of at least 40 nucleotides. For other definitions of high stringency conditions, see F. Ausubel et al., Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6, John Wiley and Sons, New York, N.Y., 1994, hereby incorporated by reference.
By xe2x80x9canalyzingxe2x80x9d or xe2x80x9canalysisxe2x80x9d is meant subjecting a methionine synthase nucleic acid or methionine synthase polypeptide to a test procedure that allows the determination of whether a methionine synthase gene is wild-type or mutant. For example, one could analyze the methionine synthase genes of an animal by amplifying genomic DNA using the polymerase chain reaction, and then determining the DNA sequence of the amplified DNA.
By xe2x80x9cprobexe2x80x9d or xe2x80x9cprimerxe2x80x9d is meant a single- or double-stranded DNA or RNA molecule of defined sequence that can base pair to a second DNA or RNA molecule that contains a complementary sequence (the xe2x80x9ctargetxe2x80x9d). The stability of the resulting hybrid depends upon the extent of the base pairing that occurs. The extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules, and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art. Probes or primers specific for methionine synthase nucleic acid preferably will have at least 35% sequence identity, more preferably at least 45-55% sequence identity, still more preferably at least 60-75% sequence identity, still more preferably at least 80-90% sequence identity, and most preferably 100% sequence identity. Probes may be detectably-labelled, either radioactively, or non-radioactively, by methods well-known to those skilled in the art. Probes are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA).
By xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d means a carrier which is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington""s Pharmaceutical Sciences, (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative nucleotide substitutions typically include substitutions which generate changes within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is a methionine synthase polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure methionine synthase polypeptide may be obtained, for example, by extraction from a natural source (e.g., a fibroblast or liver cell) by expression of a recombinant nucleic acid encoding a methionine synthase polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides not only includes those derived from eukaryotic organisms but also those synthesized in E. coli or other prokaryotes.
By xe2x80x9csubstantially pure DNAxe2x80x9d is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic mammals (e.g., rodents such as rats or mice) and the DNA (transgene) is inserted by artifice into the nuclear genome. Preferably the inserted DNA encodes a protein in at least some cells of the organism.
By xe2x80x9cknockout mutationxe2x80x9d is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% relative to the unmutated gene. The mutation may, without limitation, be an insertion, deletion, frameshift mutation, or a missense mutation. Preferably, the mutation is an insertion or deletion, or is a frameshift mutation that creates a stop codon.
By xe2x80x9ctransformationxe2x80x9d is meant any method for introducing foreign molecules into a cell. Lipofection, DEAE-dextran-mediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, retroviral delivery, electroporation, and biolistic transformation are just a few of the methods known to those skilled in the art which may be used. For example, biolistic transformation is a method for introducing foreign molecules into a cell using velocity driven microprojectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts which include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation may be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organelles (e.g., and mitochondria and chloroplasts), bacteria, yeast, fungi, algae, animal tissue, and cultured cells.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a methionine synthase polypeptide.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a methionine synthase polypeptide, a recombinant protein or a RNA molecule).
By xe2x80x9cpromoterxe2x80x9d is meant a minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific, temporal-specific, or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 or intron sequence regions of the native gene.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
By xe2x80x9cconserved regionxe2x80x9d is meant any stretch of six or more contiguous amino acids exhibiting at least 30%, preferably 50%, and most preferably 70% amino acid sequence identity between two or more of the methionine synthase family members, (e.g., between human and bacterial methionine synthase). Examples of conserved regions within methionine synthase are Boxes 1-4 (FIG. 1).
By xe2x80x9cdetectably-labeledxe2x80x9d is meant any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably-labeling a molecule are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as 32P or 35S) and nonradioactive labeling (e.g., chemiluminescent labeling, e.g., fluorescein labeling).
By xe2x80x9cantisensexe2x80x9d as used herein in reference to nucleic acids, is meant a nucleic acid sequence that is complementary to the coding strand of a gene, preferably, a methionine synthase gene. An antisense nucleic acid is capable of preferentially lowering the activity of a mutant methionine synthase polypeptide encoded by a mutant methionine synthase gene.
By xe2x80x9cpurified antibodyxe2x80x9d is meant antibody which is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., a methionine synthase amino-terminus-specific antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds a human methionine synthase polypeptide but that does not substantially recognize and bind other non-methionine synthase molecules in a sample, e.g., a biological sample, that naturally includes protein. A preferred antibody binds to the methionine synthase polypeptide sequence of SEQ ID NO: 2 (FIG. 3).
By xe2x80x9cneutralizing antibodiesxe2x80x9d is meant antibodies that interfere with any of the biological activities of a wild-type or mutant methionine synthase polypeptide, for example, the ability of methionine synthase to catalyze the transfer of a methyl group to homocysteine. The neutralizing antibody may reduce the ability of a methionine synthase polypeptide to catalyze the transfer preferably by 10% or more, more preferably by 25% or more, still more preferably by 50% or more, yet preferably by 70% or more, and most preferably by 90% or more. Any standard assay for the biological activity of methionine synthase, may be used to assess potentially neutralizing antibodies that are specific for methionine synthase.
By xe2x80x9cexposexe2x80x9d is meant to allow contact between an animal, cell, lysate or extract derived from a cell, or molecule derived from a cell, and a test compound.
By xe2x80x9ctreatxe2x80x9d is meant to submit or subject an animal (e.g. a human), cell, lysate or extract derived from a cell, or molecule derived from a cell to a test compound.
By xe2x80x9ctest compoundxe2x80x9d is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate an alteration in reporter gene activity or protein levels, by employing one of the assay methods described herein. Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof.
By xe2x80x9cassayingxe2x80x9d is meant analyzing the effect of a treatment, be it chemical or physical, administered to whole animals or cells derived therefrom. The material being analyzed may be an animal, a cell, a lysate or extract derived from a cell, or a molecule derived from a cell. The analysis may be for the purpose of detecting altered protein biological activity, altered protein stability, altered protein levels, altered gene expression, or altered RNA stability. The means for analyzing may include, for example, for example, the detection of the product of an enzymatic reaction, (e.g., the formation of methionine as a result of methionine synthase activity), antibody labeling, immunoprecipitation, and methods known to those skilled in the art for detecting nucleic acids.
By xe2x80x9cmodulatingxe2x80x9d is meant changing, either by decrease or increase, in biological activity.
By xe2x80x9ca decreasexe2x80x9d is meant a lowering in the level of biological activity, as measured by a lowering/increasing of: a) the formation of methionine as a result of methionine synthase activity; b) protein, as measured by ELISA; c) reporter gene activity, as measured by reporter gene assay, for example, lacZxcex2-galactosidase, green fluorescent protein, luciferase, etc.; or d) mRNA, levels of at least 30%, as measured by PCR relative to an internal control, for example, a xe2x80x9chousekeepingxe2x80x9d gene product such as xcex2-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or an externally added nucleic acid standard. In all cases, the lowering is preferably by at least 10% more preferably by at least 25%, still more preferably by at least 50%, and even more preferably by at least 70%.
By xe2x80x9can increasexe2x80x9d is meant a rise in the level of biological activity, as measured by a lowering/increasing of: a) the formation of methionine as a result of methionine synthase activity; b) protein, as measured by ELISA; c) reporter gene activity, as measured by reporter gene assay, for example, lacZxcex2-galactosidase, green fluorescent protein, luciferase, etc.; or d) mRNA, levels of at least 30%, as measured by PCR relative to an internal control, for example, a xe2x80x9chousekeepingxe2x80x9d gene product such as xcex2-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or an externally added nucleic acid standard. Preferably, the increase is by 10% or more, more preferably by 25% or more, even more preferably by 2-fold, and most preferably by at least 3-fold.
By xe2x80x9calteration in the level of gene expressionxe2x80x9d is meant a change in gene activity such that the amount of a product of the gene, i.e., mRNA or polypeptide, is increased or decreased, or that the stability of the mRNA or the polypeptide is increased or decreased.
By xe2x80x9creporter genexe2x80x9d is meant any gene which encodes a product whose expression is detectable and/or quantitatable by immunological, chemical, biochemical or biological assays. A reporter gene product may, for example, have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZxcex2-galactosidase, luciferase, chloramphenicol acetyltransferase), toxicity (e.g., ricin A), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably labelled antibody). It is understood that any engineered variants of reporter genes, which are readily available to one skilled in the art, are also included, without restriction, in the forgoing definition.
By xe2x80x9cproteinxe2x80x9d or xe2x80x9cpolypeptidexe2x80x9d or xe2x80x9cpolypeptide fragmentxe2x80x9d is meant any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide.
By xe2x80x9cmissense mutationxe2x80x9d is meant the substitution of one purine or pyrimidine base (i.e. A, T, G, or C) by another within a nucleic acid sequence, such that the resulting new codon encodes an amino acid distinct from the amino acid originally encoded by the reference (e.g. wild-type) codon.
By xe2x80x9cdeletion mutationxe2x80x9d is meant the deletion of at least one nucleotide within a polynucleotide coding sequence. A deletion mutation alters the reading frame of a coding region unless the deletion consists of one or more contiguous 3-nucleotide stretches (i.e. xe2x80x9ccodonsxe2x80x9d). Deletion of a codon from a nucleotide coding region results in the deletion of an amino acid from the resulting polypeptide.
By xe2x80x9cframeshift mutationxe2x80x9d is meant the insertion or deletion of at least one nucleotide within a polynucleotide coding sequence. A frameshift mutation alters the codon reading frame at and/or downstream from the mutation site. Such a mutation results either in the substitution of the encoded wild-type amino acid sequence by a novel amino acid sequence, or a premature termination of the encoded polypeptide due to the creation of a stop codon, or both.